Partridge C A, Phillips P G, Niedbala M J, Jeffrey J J
Department of Biochemistry and Molecular Biology, Albany Medical College, New York, USA.
Am J Physiol. 1997 May;272(5 Pt 1):L813-22. doi: 10.1152/ajplung.1997.272.5.L813.
The cell-surface localization and site of activation of type IV collagenases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonary microvascular endothelial (BPMVE) cells was examined. Sucrose density centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalization with beta 1-integrin, indicating incorporation into the focal contacts. The focal contacts were extracted with saponin, and associated proteolytic activity was examined by zymography. The focal contacts contained latent MMP-2, and stimulation of the cells with cytochalasin D or with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-methylxanthine increased both latent and activated MMP-9 in the focal contacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact extracts was derived from previously secreted enzyme. The activated metalloproteinase degraded extracellular matrix collagens and was inhibited by 1,10-phenanthroline. These findings indicate that endothelial cells release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.
研究了牛肺微血管内皮（BPMVE）细胞中IV型胶原酶/明胶酶（基质金属蛋白酶，MMP）的细胞表面定位及激活位点。质膜的蔗糖密度离心及全细胞免疫荧光染色表明，72 kDa（MMP-2）和96 kDa（MMP-9）的IV型胶原酶/明胶酶与质膜相关。用罗丹明标记的MMP-9孵育BPMVE细胞，结果显示其与β1整合素共定位，表明其掺入黏着斑。用皂苷提取黏着斑，并通过酶谱法检测相关的蛋白水解活性。黏着斑含有潜伏的MMP-2，用细胞松弛素D或用8-溴腺苷3'，5'-环磷酸与3-异丁基-1-甲基黄嘌呤刺激细胞，可增加黏着斑中潜伏的和激活的MMP-9。在无血清培养基中添加这些刺激物不会产生这种效果，这表明黏着斑提取物中的MMP-9源自先前分泌的酶。激活的金属蛋白酶可降解细胞外基质胶原蛋白，并被1,10-菲咯啉抑制。这些发现表明，内皮细胞将MMP释放到细胞外环境中，然后在黏着斑处从培养基中浓缩并激活MMP-9。
